It is now well established that the potential of Magnetic Resonance Imaging (MRI) procedures can be further enhanced when this diagnostic modality is applied in conjunction with the administration of contrast agents (CAs), i.e. chemicals able to promote marked changes in the relaxation rates of the tissue protons. According to the major effects they produce on images, CAs are classified as positive or negative agents. The positive CAs are represented by paramagnetic complexes, mostly containing Gd(III) or Mn(II) ions, which affect the relaxation rates of the bulk water through the exchange of the water molecules in their coordination spheres (Caravan P, et al. Chem Rev 1999, 99:2293-2352; the Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging. Chichester, UK: John Wiley & Sons; 2001. p 45-120). Their effect is similar on T1 and T2 but, being T1 usually significantly longer than T2 in most biological tissues, their effect is more often exploited in T1-weighted images, thus resulting in brighter spots in the images.
On the contrary, negative CAs are used to shorten T2, leading to an improved contrast by reducing the water signal in T2-weighted images.
Furthermore, it was early reported that chemicals containing mobile protons may act as T2-agents through the reduction of the water proton relaxation time via exchange processes (Aime S et al., Invest Radiol 1988; 23(Suppl 1):S267-S2706).
A different way to efficiently reduce water signal occurs when a proper radiofrequency pulse (rf) is applied at the resonance frequency of an exchangeable proton saturating it. This results in a net decrease of the bulk water signal intensity owing to a saturation transfer effect.
This alternative MRI contrast-enhancing technique is named Chemical Exchange Dependent Saturation Transfer (CEDST or, more commonly, CEST) (Balaban R S.: Young I R, editor. Methods in Biomedical Magnetic Resonance Imaging and Spectroscopy. Chichester, UK: John Wiley & Sons; 2000. Vol. 1. p 661-6667).
Suitable contrast agents for this technique include at least one chemically exchangeable proton.
The efficacy of most prior art contrast agents, either the conventional T1 and T2-reducing MRI contrast agents or the CEST agents, is related to the different cellular uptake of the administered complex compound or to the different distribution thereof through the extracellular spaces of the targeted organ or tissue. No contrast is detectable if the uptake between the target and the surrounding tissue is similar.
Moreover, the prior art contrast agents are expressly addressed to the production of images of the targeted tissue or organ and, generally, are unable to act as reporter of specific physical or chemical parameters of the examined tissue which could represent a quantitative assessment of a physio/pathological state.
Otherwise, WO 00/66180 discloses a method for enhancing the contrast of MRI images which comprise the administration of a CEST MRI contrast agent including at least one chemical group endowed with appropriate proton exchange and chemical shift properties to function effectively for performing CEST MRI analyses in vivo as well as for the determination of physical or chemical parameters such as pH and temperature both in vivo and in vitro. A ratiometric method for the pH measurement which is independent on the contrast agent concentration is also disclosed.
All the agents disclosed by Balaban and co-workers as useful to practice the claimed method are diamagnetic organic molecules having OH or NH exchangeable protons.
In general, the mobile protons of a contrast agent for a CEST application must possess a fast exchange rate (kex) with water protons, but slower than the coalescence condition, wherein this condition is suitably reached when kexΔv˜1/2π, where Δv is the chemical shift separation in Hz between the two exchanging pools. In this context, larger Δv values enable the exploitation of higher kex values, thus resulting in an enhanced CEST effect.
The diamagnetic systems claimed by Balaban are advantageously endowed with adequately short relaxation rates, but the chemical shifts separation from their NH or OH exchangeable protons signals and the bulk water signal is only within 1-5 ppm. So, the saturation of these mobile protons, avoiding the saturation of the bulk water or protein bound water, could actually present a considerable difficulty. Beside the small Δv values, a further limit of such diamagnetic agents is represented by the high concentration thereof which is usually required to generate a sufficiently large CEST effect, resulting in a high probability of toxic or physiological effect in vivo.
WO 02/43775 discloses paramagnetic metal ion-based macrocyclic CEST contrast agents which comprises a tetraazacyclododecane ligand wherein pendent arms includes amide groups, a paramagnetic metal ion coordinated to the ligand and a water molecule associated with it. Said agents are reported to be useful for producing image contrast based on a magnetization transfer mechanism.
The specification cites the effect of the pH on the residence lifetime at 298 K, τM298, (τM=1/kex) for protons associated with the amides in the pendent arms and the pH dependence of the Magnetization Transfer effect obtained while saturating two magnetically different exchangeable protons associated to the same amide group of one of claimed compounds (FIG. 25 and experiment 13, respectively). The specification, however, fails to teach or even suggest the applicability of the pH effect on the magnetization transfer to the whole class of claimed agents. Moreover, either the specification or the experiment 13 fail to teach or to suggest the possible use of the claimed compounds in a method of general applicability for the determination of a physical or chemical parameter of diagnostic interest in a human or animal body organ, fluid or tissue; even less in a method wherein said determination is obtained independently on the local contrast agent concentration. At the same time, WO 02/43775 specification fails to teach how said determination may be carried out.